PROJECT SUMMARY/ABSTRACT The mouse molar provides an excellent model system for studying epithelial and mesenchymal tissue interactions during organogenesis because of its relatively simple physiology and easy access. Using mouse genetics, scientists have uncovered a number of signaling pathways that regulate tooth formation. Depending on the range, duration, and intensity of the signal, intracellular cascades can initiate different cellular programs for proliferation, differentiation, migration, and survival. The Sprouty family of genes is essential for fine-tuning receptor-tyrosine-kinase signaling initiated by FGFs during tooth formation. While it is known that Sprouty genes act as negative feedback regulators of the FGF-RAS/MAPK and ?RAS/PI3K pathways, the molecular mechanisms that differentially induce Sprouty gene expression and direct divergent cellular responses remain unclear. Mice carrying single and various combinations of Sprouty mutant alleles have supernumerary teeth and display abnormalities in tooth size, shape, and micro-structure. Therefore, I hypothesize that SPRY1 and SPRY2 fine-tune FGF signaling through specific signaling cascades to control proliferation and differentiation of the dental epithelium and mesenchyme, subsequently regulating tooth patterning and morphogenesis. Preliminary results show that Spry1 and Spry2 are expressed in both distinct and overlapping regions of the epithelium and mesenchyme, and loss of function results in the formation of a supernumerary bud, lingual to the first molar. Cultured E14.5 dental germs under kidney capsule in adult nude mice for four weeks demonstrated the potential of the supernumerary buds to form mineralized teeth. Precise characterization of the expression of Sprouty genes and how they alter cell transcriptome and behavior will addressed in Aim 1 using immunostaining, static and dynamic histomorphometrical analyses, and single cell RNA-seq. In Aim 2, the proposed experiments will determine the precise point in the cascade at which SPRY1 and SPRY2 intercept FGF-RAS signaling in molar development. Inhibitors targeting the identified hyper-phosphorylated kinases will be utilized in attempt to rescue the mutant phenotype. This proposed work explores the mechanism whereby Sprouty proteins control tissue- specific interactions during development of the mouse molar. Elucidation of the regulatory network and functional relationship between SPRY1 and SPRY2 and FGF signaling in vivo will advance our knowledge of how Sprouty proteins control tooth organogenesis and lead to novel molecular-based therapies for detection and treatment and, in the long run, to bioengineering of teeth.